RNAScope – a new FISH in the sea

I recently started a collaboration that involves the use of the RNAScope method. So here’s a short overview of the method.

FISH is a very useful method to observe and quantify specific RNA species in situ. Yet, a major issue is the signal to noise ratio. Single probes can attach non-specifically in the cell and create background fluorescence. One way to overcome this background is to use multiple probes against the same RNA, but at different locations along the RNA. The more probes specifically attach to the RNA, the better is the signal to noise ratio. However, I know from experience that it is still fairly difficult to distinguish true RNA spots from background signal.

RNAScope, developed by a team from Advanced Cell Diagnostics, tries to overcome this problem from another angle. Instead of having multiple labeled probes against the target RNA, they produce two unlabeled tandem probes. These probes contain a short complementary region (18-25 bases), a spacer sequence and a 14-base tail sequence.

Schematic of the RNAscope assay procedure. In step 1, cells or tissues are fixed and permeabilized to allow for target probe access. In step 2, target RNA-specific oligonucleotide probes (Z) are hybridized in pairs (ZZ) to multiple RNA targets. In step 3, multiple signal amplification molecules are hybridized, each recognizing a specific target probe, and each unique label probe is conjugated to a different fluorophore or enzyme. In step 4, signals are detected using a fluorescent microscope. Source: Wang et al. (2012) J. Molec. Diag. 14:22.

After hybridization with the target probes, comes a second hybridization step with a pre-amplifier probe. This is a long probe that contains a complementary sequence to the 28 bases of the two target probes tails (14+14). So, only when the two target tails hybridize one next to the other the pre-amplifier will hybridize. The pre-amplifier contains 20 binding sites for an amplifier probe which in turn contains 20 binding sites for the labeled probe. Thus, for each target probe pair, we get 20×20=400 labeled probes.

That is a large amplification. They suggest having ~20 target probe pairs per RNA. So each RNA is amplified ~8000fold (assuming 100% efficiency of hybridization) over the background of single labeled probes.

In their paper, they show convincing images of their negative controls (single target probe compared to no-probe). However, they do not supply any statistics (i.e. how many cells/fields they observed, how many biological repeats, are there cells with some detectable spots?)

Validation of RNAscope. HeLa cells were hybridized with either the full set of probes to 18S rRNA, the left half of the set, or the right half of the set (as shown in the schematic along the top). A no-probe control was performed in parallel as an indicator of background staining. Cells were counterstained with DAPI (blue), which masks nucleolar 18S RNA. Source: Wang et al. (2012) J. Molec. Diag. 14:22.

The main object of developing this method, they claim, is to have a good tool for molecular pathology, i.e. – a good method to examine RNA in situ in pathological tissue samples. In their paper, they go on to show RNAScoping of specific mRNAs in cells cultures and in tissue samples. It looks very good.

My associates are going to try this method with my cells within a couple of weeks. We’ll see if it works as good as they claim.

13 responses to “RNAScope – a new FISH in the sea”

Hi,
I am also interested to know how well this method worked for your colleagues. Also, if you heard of anyone using this method with FACS instead of microscopy, I would appreciate it if you can tell me.
Thanks,
Ilya.

yep, I was surprised too it didn’t work. I should mention that they also tried for GFP and it seemed to have worked.
Other people use the same approach, they just don’t call it RNAScope: http://www.ncbi.nlm.nih.gov/pubmed/24097269

Hello Galicolagfb. Thanks for the information (and the blog in general). I am extremely interested in flow cytometric applications (and surely not alone in this…). Aren’t RNAScope and bDNA essentially the same? I have been looking for a way to make the reagents myself to get around paying exorbitant sums to the commercial companies (we are a small lab in a wee bit of a bind…) but all seems to be covered by tons of patents and the original literature is quite old: so, long story short, is there a general protocol for the construction of the detection systems (i.e. the branching)?

Hi Paolo,
yes, RNAScope and bDNA are essentially the same. However, there is one major difference: in RNAScope, the 2nd probe needs to bind TWO adjacent 1st-level probes (the RNA specific probes), which should give less background than bDNA.
I haven’t designed the probes myself – I just sent my cells to the people at BD to see if they can detect my mRNA.
For some reason, it didn’t work. I don’t know why, they just say they don’t get any signal. As of now, I’m not doing anything in that direction anymore.
So, in short – I do not have any protocol.

Hi Paolo,
I was interested in this technology for flow cytometry uses as well. The QuantiGene FlowRNA product looks promising to me: http://www.ebioscience.com/application/flowrna.htm
If you eventually decide to use a commercial product, it would be nice if you can update us here with your experience.
Best,
Ilya.

Hi Paolo,
Thanks. It sounds nice, but I guess we’ll have to wait for the paper.

Anyway, I think that I have an explanation as to why the guys from BD that I collaborated with couldn’t detect my mRNA. My mRNA encodes a cytoskeletal protein. Now, their protocol begins with harvesting the cells and centrifuging them prior to fixation. In case of adherent cells, like mine, this totally alters the cell shape (from cells that are spread out, they become round and small) and hence the cytoskeleton. This cytoskeleton remodeling is accompanied, I think, by massive degradation* of the mRNA – and I have preliminary data that suggests that this may be the case. But I still need to do a controlled experiment to test that theory.

* Instead of degradation, it could be aggregation into stress granules, which may mask the mRNA, similar to what was recently shown for dendritic mRNAs: http://wp.me/p2jizY-9F

The fate of the messenger is pre-determined

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